Genomic Diversity and Zoonotic Potential of Brucella neotomae

After reports in 2017 of Brucella neotomae infections among humans in Costa Rica, we sequenced 12 strains isolated from rodents during 1955–1964 from Utah, USA. We observed an exact strain match between the human isolates and 1 Utah isolate. Independent confirmation is required to clarify B. neotomae zoonotic potential.

We generated a maximum parsimony tree from the 149 SNPs identified among the 27 B. neotomae sequence datasets, including 15 public and 12 newly sequenced WGS datasets (Figure 1; Appendix Tables 1, 2).The whole-genome SNP (wgSNP) genotype of the most recent common ancestor (MRCA) of known B. neotomae lineages descends into 2 groups (Figure 1): the group containing type strain 5K33 corresponds to sequence type (ST) 22 in the Brucella multilocus sequence typing scheme MLST21, the other to ST120 (7).The limited available information about the sampling site of each strain from rodents in the Great Salt Lake Desert in Utah, USA, is consistent with congruence between B. neotomae phylogeny and the geography of the Great Salt Lake region, but further data are needed to robustly test this association (Appendix).
We show a different representation of the wgSNP phylogenetic analysis after removal of duplicates and of 1 dataset with relatively lower coverage (Figure 2; GenBank accession no.GCA_900446105 from strain 6D152).Because we removed the WGS datasets with partial coverage, the new tree contained 205 SNPs.The distances from MRCA to tips were similar: maximum 76 SNPs (to strain 7E1260) and minimum 56 SNPs (to strain APHA#65-197).The 3 whole-genome datasets from Costa Rica, including the human isolates bneohCR1 and bneohCR2 and the isolate babohCR62 entered as B. abortus in the European Nucleotide Archive database, remained identical in wgSNP genotype to strain 5G239 (BCCN#R34) in spite of the increased resolution.We still observed a coincident wgSNP genotype when we considered only these 4 strains, in sharp contrast with a report of human cases that indicated the corresponding genomes differed by 164 SNPs (4).

Conclusions
Our findings demonstrate that the strains isolated during 1955-1964 in the Great Salt Lake Desert in Utah display notable intraspecies genetic diversity despite being isolated from a geographically limited location, within a limited time frame, and from the same host species.In contrast, the datasets from wgSNP analysis of isolates from Costa Rica were identical despite having been isolated 4 years apart and in different areas of Costa Rica (5).Of note, datasets from analysis of isolates from Costa Rica were identical to data from 1 B. neotomae strain, 5G239, from the Great Salt Lake region.Finding an identical genotype in human cases from Costa Rica >3,000 km and >50 years apart in a different species from the Great Salt Lake discovery is remarkable in light of the diversity of strains noted in the geographically limited location in Utah and reported absence of rats of genus Neotoma in Costa Rica (5).Full understanding of the zoonotic potential of B. neotomae requires further exploration, including additional sampling of rodents and human cases in the US Southwest and Central America.

About the Author
Dr. Vergnaud is a research associate at University Paris-Saclay, Gif-sur-Yvette, France.His research interests currently focus on the evolution and phylogeography of a number of dangerous bacterial pathogens.F or the sesquipedalian term Haematospirillum, Haema is derived from the Greek haima, meaning blood.Spirillum is derived from Medieval Latin in the mid-13th century Latin (spiralis), French in the 1550s (spiral), and Greek (speira).All suggest a winding or coil.A New Latin reference book entry in 1875 implied a little coil (Figure 1).Isolated from human blood, Haematospirillum jordaniae was reported as a novel genus and species in 2016 by Centers for Disease Control and Prevention (CDC) scientist Ben W. Humrighouse and his laboratory team, which included Jean G. Jordan, a microbiologist (Figure 2).This gram-negative bacterium was isolated 14 times in 10 states during 2003-2012 before its identification in 2016.
H. jordaniae was previously considered an environmental bacterium with limited pathogenicity, but increasing numbers of isolates indicated a possible emerging pathogen.All cases occurred in male patients, and the pathogen showed a predilection for infecting lower leg injuries.In 2018, Hovan and Hollinger reported a case of infection in a Delaware man who, in 2016, had sepsis from a lower leg wound.The organism isolated was identified at the CDC Special Bacteriology Reference Laboratory (SBRL) in the Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases.

Figure 1 .
Figure 1.Brucella neotomae phylogeny.Maximum parsimony tree was derived from wgSNP data.We investigated 27 datasets and identified 149 SNPs; tree size is 151 substitutions (homoplasy 1.5%).Circles are colored according to primary strain identifier; red indicates the 3 datasets from Costa Rica.Circles are labeled with an accession number or collection strain identifier (Brucella Culture Collection Nouzilly [BCCN]) or Animal and Plant Health Agency [APHA] Weybridge collections).Branch lengths >1 substitution are indicated.Black star shows the position of the hypothetical MRCA.Box indicates the 2 MLST21 STs.MLST, multilocus sequence typing; MRCA, most recent common ancestor; SNP, single-nucleotide polymorphism; ST, sequence type; wgSNP, whole-genome singlenucleotide polymorphism.
Work conducted at INRAE and University Paris-Saclay was supported in part by ANR (Agence Nationale de la Recherche; French National Research Agency) ASTRIDmaturation project ANR-14-ASMA-0002 (Global surveillance of infectious agents: molecular typing kits and databases-MicroType). Work conducted at UK APHA was supported in part by the One Health EJP project (JRP-17-IDEMBRU: Identification of emerging Brucella species: new threats for human and animals; grant agreement no.773830).

Figure 2 .
Figure 2. Rooted Brucella neotomae phylogeny of 16 selected datasets.Maximum parsimony tree was derived from wgSNP data; 205 SNPs in tree, tree size 207 substitutions (homoplasy 1%).Branch lengths >1 substitution are indicated.Circles are colored according to primary strain identity; red indicates the 3 datasets from Costa Rica.Circles are labeled with an accession number or collection strain identifier (Brucella Culture Collection Nouzilly [BCCN] or Animal and Plant Health Agency [APHA] Weybridge collections).Primary strain identifier is indicated in brackets when available.ENA, European Nucleotide Archive; SNP, single-nucleotide polymorphism; wgSNP, whole-genome SNP.